Adrienne W. Paton

A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans.

Gastrointestinal disease caused by Shiga toxin-producing bacteria (such as Escherichia coli O157:H7 and Shigella dysenteriae) is often complicated by life-threatening toxin-induced systemic sequelae, including hemolytic–uremic syndrome. Such infections can now be diagnosed very early in the course of the disease, but at present no effective therapeutic intervention is possible. Here, we constructed a recombinant bacterium that displayed a Shiga toxin receptor mimic on its surface, and it adsorbed and neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the recombinant bacterium completely protected mice from challenge with an otherwise 100%-fatal dose of Shiga toxigenic E. coli. Thus, the bacterium shows great promise as a ‘probiotic’ treatment for Shiga toxigenic E. coli infections and dysentery.

Heterogeneity of the amino-acid sequences of Escherichia coli shiga-like toxin type-I operons

PCR was used to amplify approx. 1470-bp segments of DNA containing complete Shiga-like toxin type I (sltI) operons from Escherichia coli strains belonging to serotypes O48:H21, O111:H- and OX3:H8. These fragments were cloned and DNA sequence analysis identified several variations, as compared with published sltI sequences. All three sltI genes analysed were more closely related to Shiga toxin-encoding genes (sht) of Shigella dysenteriae type 1, than to previously published E. coli phage-encoded sltI genes. The greatest deviation in deduced amino acid (aa) sequence was observed in the SltI protein from the OX3:H8 strain, which differed from the phage 933J-encoded SltI by 9 aa in the A subunit and 3 aa in the B subunit.

Cloning and nucleotide sequence of a variant Shiga-like toxin II gene from Escherichia coli OX3:H21 isolated from a case of sudden infant death syndrome☆

Escherichia coli OX3:H21 expressing a toxin related to Shiga-like toxin (SLT) was isolated from the small bowel contents of a case of Sudden Infant Death Syndrome (SIDS). This strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-related gene was located on a 4.6 kb PstI fragment, which was cloned into E. coli JM109 in both orientations, using the vector pUC19, to generate plasmids pJCP501 and pJCP502. JM109 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells. Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from, previously reported variants of Shiga-like toxin type II, produced by E. coli from both human and animal sources. The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-II variant. The B subunit was less similar, exhibiting 88.6 and 88.8% homology to the related gene at the DNA and amino acid level, respectively.

Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H−

PCR amplification was used to screen faecal isolates of Escherichia coli from a 12-month-old boy with haemolytic uraemic syndrome for the presence of Shiga-like toxin (SLT)-encoding genes. One isolate, belonging to serotype O111:H-, was positive for SLT-I by this method. UV induction indicated that the strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-I gene was located on an 8.5-kb EcoRI fragment. SLT-I was further localized to within a 3.0-kb SphI-EcoRI fragment. A separate subclone contained a 3.75-kb HindIII fragment, 1.18 kb of which was common to both. Nucleotide sequence analysis of derivatives of these clones revealed that the SLT-I A subunit gene from E. coli O111:H- differed from the previously published sequences for SLT-I by 5 bp [resulting in two amino acid (aa) changes]. It was more closely related to the gene encoding the A subunit of the Shiga toxin from Shigella dysenteriae type 1, from which it differed by 3 bp (resulting in one aa change). The DNA sequence of the B subunit-encoding gene was identical to that of the other two toxins. The region of DNA upstream from the SLT-I of E. coli O111:H- contained an IS element, as well as a region with strong homology to a portion of the genome of bacteriophage lambda.

Characterization of IS1203, an insertion sequence in Escherichia coli 0111:H−

The complete nucleotide (nt) sequence of IS1203 from Escherichia coli O111:H- strain PH has been determined. IS1203 is 1312-nt long, with imperfect 26-bp terminal inverted repeats. The two major ORFs in IS1203 encode polypeptides of 12.7 and 33.7 kDa, the latter being the putative transposase. IS1203 is closely related to IS629 of Shigella sonnei and IS3411 of E. coli. At least twelve copies of IS1203 were found in the genome of E. coli O111:H- strain PH.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.